Adenosine A2A receptors are required for glutamate mGluR5‐ and dopamine D1 receptor‐evoked ERK1/2 phosphorylation in rat hippocampus: involvement of NMDA receptor

Interaction between mGluR5 and NMDA receptors (NMDAR ) is vital for synaptic plasticity and cognition. We recently demonstrated that stimulation of mGluR5 enhances NMDAR responses in hippocampus by phosphorylating NR2B(Tyr1472) subunit, and this reaction was enabled by adenosine A_2A receptors (A_2AR) (J Neurochem, 135, 2015, 714). In this study, by using in vitro phosphorylation and western blot analysis in hippocampal slices of male Wistar rats, we show that mGluR5 stimulation or mGluR5/NMDAR s co‐stimulation synergistically activate ERK 1/2 signaling leading to c‐Fos expression. Interestingly, both reactions are under the permissive control of endogenous adenosine acting through A_2ARs. Moreover, mGluR5‐mediated ERK 1/2 phosphorylation depends on NMDAR , which however exhibits a metabotropic way of function, since no ion influx through its ion channel is required. Furthermore, our results demonstrate that mGluR5 and mGluR5/NMDAR ‐evoked ERK 1/2 activation correlates well with the mGluR5/NMDAR ‐evoked NR2B(Tyr1472) phosphorylation, since both phenomena coincide temporally, are Src dependent, and are both enabled by A_2ARs. This indicates a functional involvement of NR2B(Tyr1472) phosphorylation in the ERK 1/2 activation. Our biochemical results are supported by electrophysiological data showing that in CA 1 region of hippocampus, the theta burst stimulation (TBS)‐induced long‐term potentiation coincides temporally with an increase in ERK 1/2 activation and both phenomena are dependent on the tripartite A_2A, mGlu5, and NMDAR s. Furthermore, we show that the dopamine D1 receptors evoked ERK 1/2 activation as well as the NR2B(Tyr1472) phosphorylation are also regulated by endogenous adenosine and A_2ARs. In conclusion, our results highlight the A_2ARs as a crucial regulator not only for NMDAR responses, but also for regulating ERK 1/2 signaling and its downstream pathways, leading to gene expression, synaptic plasticity, and memory consolidation.

     

Thymus vulgaris alleviates UVB irradiation induced skin damage via inhibition of MAPK/AP‐1 and activation of Nrf2‐ARE antioxidant system

Solar ultraviolet (UV) radiation‐induced reactive oxidative species is mainly responsible for the development of photoageing. Rosmarinic acid was one of the main bioactive components detected in Thymus vulgaris (TV) we extracted. In this study, UVB‐induced skin damages have been shown to be ameliorated by treatment with TV in hairless mice (HR‐1) skin, demonstrated by decreased matrix metalloproteinases (MMPs) and increased collagen production. However, the underlying molecular mechanism on which TV acted was unclear. We examined the photoprotective effects of TV against UVB and elucidated the molecular mechanism in normal human dermal fibroblasts. Thymus vulgaris remarkably prevented the UVB‐induced reactive oxygen species and lactate dehydrogenase. Dose‐dependent increase in glutathione, NAD(P)H: quinone oxidoreductase1 and heme oxygenase‐1, by TV was confirmed by increased nuclear accumulation of Nrf2. Furthermore, 5‐Methoxyindole‐2‐carboxylic acid was introduced as a specific inhibitor of dihydrolipoyl dehydrogenase (DLD). We demonstrated that Nrf2 expression was regulated by DLD, which was a tricarboxylic acid cycle‐associated protein that decreased after UVB exposure. Besides, TV significantly diminished UVB induced phosphorylation of mitogen activated protein kinases pathway, containing extracellular signal‐regulated kinase, Jun N‐terminal kinase and p38, which consequently reduced phosphorylated c‐fos and c‐jun. Our results suggest that TV is a potential botanical agent for use against UV radiation‐induced oxidative stress mediated skin damages.     

ATM/Wip1 activities at chromatin control Plk1 re‐activation to determine G2 checkpoint duration

After DNA damage, the cell cycle is arrested to avoid propagation of mutations. Arrest in G2 phase is initiated by ATM‐/ATR‐dependent signaling that inhibits mitosis‐promoting kinases such as Plk1. At the same time, Plk1 can counteract ATR‐dependent signaling and is required for eventual resumption of the cell cycle. However, what determines when Plk1 activity can resume remains unclear. Here, we use FRET‐based reporters to show that a global spread of ATM activity on chromatin and phosphorylation of ATM targets including KAP1 control Plk1 re‐activation. These phosphorylations are rapidly counteracted by the chromatin‐bound phosphatase Wip1, allowing cell cycle restart despite persistent ATM activity present at DNA lesions. Combining experimental data and mathematical modeling, we propose a model for how the minimal duration of cell cycle arrest is controlled. Our model shows how cell cycle restart can occur before completion of DNA repair and suggests a mechanism for checkpoint adaptation in human cells.     

Genetic removal of eIF2α kinase PERK in mice enables hippocampal L‐LTP independent of mTORC1 activity

Characterization of the molecular signaling pathways underlying protein synthesis‐dependent forms of synaptic plasticity, such as late long‐term potentiation (L‐LTP ), can provide insights not only into memory expression/maintenance under physiological conditions but also potential mechanisms associated with the pathogenesis of memory disorders. Here, we report in mice that L‐LTP failure induced by the mammalian (mechanistic) target of rapamycin complex 1 (mTORC 1) inhibitor rapamycin is reversed by brain‐specific genetic deletion of PKR ‐like ER kinase, PERK (PERK KO ), a kinase for eukaryotic initiation factor 2α (eIF 2α). In contrast, genetic removal of general control non‐derepressible‐2, GCN 2 (GCN 2 KO ), another eIF 2α kinase, or treatment of hippocampal slices with the PERK inhibitor GSK 2606414, does not rescue rapamycin‐induced L‐LTP failure, suggesting mechanisms independent of eIF 2α phosphorylation. Moreover, we demonstrate that phosphorylation of eukaryotic elongation factor 2 (eEF 2) is significantly decreased in PERK KO mice but unaltered in GCN 2 KO mice or slices treated with the PERK inhibitor. Reduction in eEF 2 phosphorylation results in increased general protein synthesis, and thus could contribute to the mTORC 1‐independent L‐LTP in PERK KO mice. We further performed experiments on mutant mice with genetic removal of eEF 2K (eEF 2K KO ), the only known kinase for eEF 2, and found that L‐LTP in eEF 2K KO mice is insensitive to rapamycin. These data, for the first time, connect reduction in PERK activity with the regulation of translation elongation in enabling L‐LTP independent of mTORC 1. Thus, our findings indicate previously unrecognized levels of complexity in the regulation of protein synthesis‐dependent synaptic plasticity.

Read the Editorial Highlight for this article on page 119 . Cover Image for this issue: doi: 10.1111/jnc.14185 .

     

Prostaglandin I2 upregulates the expression of anterior pharynx‐defective‐1α and anterior pharynx‐defective‐1β in amyloid precursor protein/presenilin 1 transgenic mice

Cyclooxygenase‐2 (COX‐2) has been recently identified to be involved in the pathogenesis of Alzheimer's disease (AD). Yet, the role of an important COX‐2 metabolic product, prostaglandin (PG) I₂, in the pathogenesis of AD remains unknown. Using human‐ and mouse‐derived neuronal cells as well as amyloid precursor protein/presenilin 1 (APP/PS1) transgenic mice as model systems, we elucidated the mechanism of anterior pharynx‐defective (APH)‐1α and pharynx‐defective‐1β induction. In particular, we found that PGI₂ production increased during the course of AD development. Then, PGI₂ accumulation in neuronal cells activates PKA/CREB and JNK/c‐Jun signaling pathways by phosphorylation, which results in APH‐1α/1β expression. As PGI₂ is an important metabolic by‐product of COX‐2, its suppression by NS398 treatment decreases the expression of APH‐1α/1β in neuronal cells and APP/PS1 mice. More importantly, β‐amyloid protein (Aβ) oligomers in the cerebrospinal fluid (CSF) of APP/PS1 mice are critical for stimulating the expression of APH‐1α/1β, which was blocked by NS398 incubation. Finally, the induction of APH‐1α/1β was confirmed in the brains of patients with AD. Thus, these findings not only provide novel insights into the mechanism of PGI₂‐induced AD progression but also are instrumental for improving clinical therapies to combat AD.     

Drosophila insulin‐like peptide dilp1 increases lifespan and glucagon‐like Akh expression epistatic to dilp2

Insulin/IGF signaling (IIS) regulates essential processes including development, metabolism, and aging. The Drosophila genome encodes eight insulin/IGF‐like peptide (dilp) paralogs, including tandem‐encoded dilp1 and dilp2. Many reports show that longevity is increased by manipulations that decrease DILP2 levels. It has been shown that dilp1 is expressed primarily in pupal stages, but also during adult reproductive diapause. Here, we find that dilp1 is also highly expressed in adult dilp2 mutants under nondiapause conditions. The inverse expression of dilp1 and dilp2 suggests these genes interact to regulate aging. Here, we study dilp1 and dilp2 single and double mutants to describe epistatic and synergistic interactions affecting longevity, metabolism, and adipokinetic hormone (AKH), the functional homolog of glucagon. Mutants of dilp2 extend lifespan and increase Akh mRNA and protein in a dilp1‐dependent manner. Loss of dilp1 alone has no impact on these traits, whereas transgene expression of dilp1 increases lifespan in dilp1 ? dilp2 double mutants. On the other hand, dilp1 and dilp2 redundantly or synergistically interact to control circulating sugar, starvation resistance, and compensatory dilp5 expression. These interactions do not correlate with patterns for how dilp1 and dilp2 affect longevity and AKH. Thus, repression or loss of dilp2 slows aging because its depletion induces dilp1, which acts as a pro‐longevity factor. Likewise, dilp2 regulates Akh through epistatic interaction with dilp1. Akh and glycogen affect aging in Caenorhabditis elegans and Drosophila. Our data suggest that dilp2 modulates lifespan in part by regulating Akh, and by repressing dilp1, which acts as a pro‐longevity insulin‐like peptide.     

Transient and rapid activation of Akt/GSK‐3β and mTORC1 signaling by D3 dopamine receptor stimulation in dorsal striatum and nucleus accumbens

D2/D3 dopamine receptors (D2R/D3R) agonists regulate Akt, but their effects display a complex time‐course. In addition, the respective roles of D2R and D3R are not defined and downstream targets remain poorly characterized, especially in vivo. These issues were addressed here for D3R. Systemic administration of quinelorane, a D2R/D3R agonist, transiently increased phosphorylation of Akt and GSK‐3β in rat nucleus accumbens and dorsal striatum with maximal effects 10 min after injection. Akt activation was associated with phosphorylation of several effectors of the mammalian target of rapamycin complex 1 (mTORC1): p70S6 kinase, ribosomal protein‐S6 (Ser240/244), and eukaryotic initiation factor‐4E binding protein‐1. The action of quinelorane was antagonized by a D2/D3R antagonist, raclopride, and the selective D3R antagonist S33084, inactive by themselves. Furthermore, no effect of quinerolane was seen in knock‐out mice lacking D3R. In drd1a‐EGFP transgenic mice, quinelorane activated Akt/GSK‐3β in both neurons expressing and lacking D1 receptor. Thus, the stimulation of D3R transiently activates the Akt/GSK‐3β pathway in the two populations of medium‐size spiny neurons of the nucleus accumbens and dorsal striatum. This effect may contribute to the influence of D3R ligands on reward, cognition, and processes disrupted in schizophrenia, drug abuse, and Parkinson's disease.     

Long non‐coding RNA NR2F1‐AS1 promoted proliferation and migration yet suppressed apoptosis of thyroid cancer cells through regulating miRNA‐338‐3p/CCND1 axis

Thyroid cancer (TC) is a prevalent endocrine malignant cancer whose pathogenic mechanism remains unclear. The aim of the study was to investigate the roles of long non‐coding RNA (lncRNA) NR2F1‐AS1/miRNA‐338‐3P/CCND1 axis in TC progression. Differentially expressed lncRNAs and mRNAs in TC tissues were screened out and visualized by R program. Relative expression of NR2F1‐AS1, miRNA‐338‐3p and cyclin D1 (CCND1) was determined by quantitative real time polymerase chain reaction. In addition, Western blot analysis was adopted for evaluation of protein expression of CCND1. Targeted relationships between NR2F1‐AS1 and miRNA‐338‐3p, as well as miRNA‐338‐3p and CCND1 were predicted using bioinformatics analysis and validated by dual‐luciferase reporter gene assay. Besides, tumour xenograft assay was adopted for verification of the role of NR2F1‐AS1 in TC in vivo. NR2F1‐AS1 and CCND1 were overexpressed, whereas miRNA‐338‐3p was down‐regulated in TC tissues and cell lines. Down‐regulation of NR2F1‐AS1 and CCND1 suppressed proliferation and migration of TC cells yet greatly enhanced cell apoptotic rate. Silence of NR2F1‐AS1 significantly suppressed TC tumorigenesis in vivo. NR2F1‐AS1 sponged miRNA‐338‐3p to up‐regulate CCND1 expression to promote TC progression. Our study demonstrated that up‐regulation of NR2F1‐AS1 accelerated TC progression through regulating miRNA‐338‐3P/CCND1 axis.     

Heterodimerization of apelin receptor and neurotensin receptor 1 induces phosphorylation of ERK1/2 and cell proliferation via Gαq‐mediated mechanism

Dimerization of G protein‐coupled receptors (GPCRs) is crucial for receptor function including agonist affinity, efficacy, trafficking and specificity of signal transduction, including G protein coupling. Emerging data suggest that the cardiovascular system is the main target of apelin, which exerts an overall neuroprotective role, and is a positive regulator of angiotensin‐converting enzyme 2 (ACE2) in heart failure. Moreover, ACE2 cleaves off C‐terminal residues of vasoactive peptides including apelin‐13, and neurotensin that activate the apelin receptor (APJ) and neurotensin receptor 1 (NTSR1) respectively, that belong to the A class of GPCRs. Therefore, based on the similar mode of modification by ACE2 at peptide level, the homology at amino acid level and the capability of forming dimers with other GPCRs, we have been suggested that APJ and NTSR1 can form a functional heterodimer. Using co‐immunoprecipitation, BRET and FRET, we provided conclusive evidence of heterodimerization between APJ and NTSR1 in a constitutive and induced form. Upon agonist stimulation, hetrodimerization enhanced ERK_1/2 activation and increased proliferation via activation of Gq α‐subunits. These novel data provide evidence for a physiological role of APJ/NTSR1 heterodimers in terms of ERK_1/2 activation and increased intracellular calcium and induced cell proliferation and provide potential new pharmaceutical targets for cardiovascular disease.     

Pleiotrophin inhibits melanogenesis via Erk1/2‐MITF signaling in normal human melanocytes

Pleiotrophin (PTN) is a secreted heparin‐binding protein that is involved in various biological functions of cell growth and differentiation. Little is known about the effects of PTN on the melanocyte function and skin pigmentation. In this study, we investigated whether PTN would affect melanogenesis. PTN was expressed in melanocytes and fibroblasts of human skin. Transfection studies revealed that PTN decreased melanogenesis, probably through MITF degradation via Erk1/2 activation in melanocytes. The inhibitory action of PTN in pigmentation was further confirmed in ex vivo cultured skin and in the melanocytes cocultured with fibroblasts. These findings suggest that PTN is a crucial factor for the regulation of melanogenesis in the skin.